Expression of protein engineered NADP plus -dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae
Expression of protein engineered NADP plus -dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae
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DOI：
10.1007/s00253-008-1649-1
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发表时间：
2008-11-01
影响因子：
5
通讯作者：
Sawayama, Shigeki
中科院分区：
文献类型：
--
作者：
Matsushika, Akinori;Watanabe, Seiya;Sawayama, Shigeki
从树干毕赤酵母（Pichia stipitis）获得的木糖还原酶（XR）和木糖醇脱氢酶（XDH）基因转化的重组酿酒酵母（Saccharomyces cerevisiae）菌株具有将木糖转化为乙醇的能力，但木糖醇的排泄不利，这可能是由于NADPH偏好的XR和NAD+依赖的XDH之间的辅因子失衡。为了减少木糖醇的形成，我们已经产生了几种XDH突变体，其具有对NADP+的辅酶特异性的逆转。在本研究中，我们构建了一套重组S。具有木糖发酵能力的酿酒酵母菌株，包括蛋白质工程改造的NADP+依赖性XDH表达菌株。通过使用名为MA-N5的菌株发现对木糖至乙醇发酵的最积极的影响，该菌株通过NADP+依赖性XDH的基因与XR和内源性木酮糖激酶基因的染色体整合构建。与表达野生型XDH的参比菌株相比，MA-N5菌株不仅发酵木糖，而且发酵含有葡萄糖和木糖的混合糖时，乙醇产量增加，木糖醇排泄减少。此外，MA-N5菌株以0.49g乙醇/g木屑的非硫酸水解产物中总消耗糖的高产率产生乙醇。结果表明，存在于木质纤维素水解液中的葡萄糖和木糖可以被该氧化还原工程菌有效地发酵。
A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD+-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP+. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP+-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP+-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.